Life Science Journal, 3 ( 1 ) ,2006 , Yin, et aI, Construction of Prokaryotic Expression Vectors Bearing S Gene

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Construction of Prokaryotic Expression Vectors

Bearing S Gene of Isolate TH-98 fromTransmissible Gastroenteritis Virus

]iechao Yin, Guangxing Li, Yijing Li, Xiaofeng Ren

Department of Veterinary, College of Veterinary Medicine, Northeast Agricultural University,

59 Mucai Street, Harbin, Heilongjiang 150030 , China

Abstract: Sa fragment containing major antigenic sites A, E, C and D of S gene from transmissible gastroenteritisvirus (TGEV) Chinese isolate TH-98 was purified with EcoRI and Kpnl from recombinant pUC-S and cloned intoprokaryotic expression vector pProExHTb. The recombinant SaPRO was identified with restriction enzyme (RE).Sa derived from recombinant SaPCI was inserted into EcoRI and Not! sites of expression vector pET30c with thesimilar DNA recombination technique. The construct designated SaPET was transformed into Escherichia coli (E.coli) EL21. Then, SaPET was digested with Xhol and ligated in order to gain the recombinant SasPET carryingE, C and D sites of S gene, which was also transformed into EL21. The acquisition was subcloned into correspond-ing sites of another prokaryotic expression vector pGEX-6P-l after digestion with EcoRI and Xho!. The verified re-combinant Sas6P-l was re- transformed into EL21. [Life Science Journal. 2006; 3 (1) : 63 - 66] (ISSN: 1097-8135) .

Keywords: TGEV; S gene; prokaryotic expression vector; construction

1 Introduction

Transmissible gastroenteritis virus (TGEV) isone of the important pathogens for virus diarrhea ofswine (Yin, 2005). Spike(S) protein is the majorimmunogen among four major structural proteins(S, M, Nand sM) of TGEV (Krempl, 1997).Particularly the 5' end half of S gene encoding themajor antigenic sites are critical for inducing neu-tralizing antibodies (Gebauer, 1991; Tuboly,1994). To gain the S protein is a prerequisite forthe diagnosis, prevention and treatment of trans-missible gastroenteritis ( TGE). The prokaryoticexpression system shows some significant advan-tages over other systems in production cost, tech-nology and cycle etc. However, the limited expres-sion capacity for foreign genes and the different ex-pression efficiency of different vectors used in thissystem are the disadvantages.

For these reasons, we constructed several re-combinant plasmids encoding the 5' end half of Sgene or major antigenic site fragment using differ-ent prokaryotic expression vectors. These plasmidshave been transformed into Escherichia coli (E.

coli). This study provided the important materialsfor S protein expression and comparison of vector

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expression efficiency.

2 Materials and Methods

2 .1 Vector, host cells, tool enzymes and primersRecombinant plasmids pUS-C, and SaPCI re-

spectively containing full-length and 5' end half ofS gene of TGEV have been constructed in our labo-ratory (Ren, 2003). Prokaryotic expression vec-tors, pProExHTb (GIBCO), pET30c (Novagen)and pGEX-6P-1 (Amersham Biosciences) werecommercially purchased. TG1, DH5o: and BL21competent cells were also commercially obtained.Tool enzymes were purchased from Takara Biotech-nology Company (Dalian, China). Primers, PS3:5' -TACAGTGAGTGACTCGAGCT-3' (1242) andPR3 :5' -GGTGTTGTTGTCCAA TGTG-3' (2408)were used for nested PCR (The figures in bracketsare the corresponding or complementary position ofS gene).2.2 Construction of expression plasmids contain-ing S gene of TGEV

Recombinant pUC-S was digested with EcoRIand Kpnl to obtain the 5' end half of S gene namedSa. Sa was ligated with vector pProExHTb digest-ed with the same enzymes and transformed intoDH5o: cells. A positive recombinant named SaPRO

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Life ScienceJournal, 3 (1 ) ,2006, Yin, et aI, Gmstruction of ProkarYJticExpression VectorsBearing S Gene

was selected in LB agar plate containing appropriateantibiotic and identified with restriction enzyme(RE). Recombinant SaPCI was digested withEcoRI and Not I, and a linearized fragment encod-ing the Sa region was cloned into pET30c vectorusing the similar method described above. A posi-tive recombinant was named SaPET after the iden-tification with RE and by nested PCR. To facilitatefurther inducible expression, SaPET was trans-formed into BL21 cells. A strain of plasmid-con-taining ccils was selected.

DNA::S1S software analysis indicated therewere two Xlw I sites located in near nucleotide 1120of S gene and in the multiple cloning sites after Safragment in SaPET. Therefore we digested SaPETwith Xho I and a resulting fragment containing vec-tor and 5' end S gene about 1100 base pair (bp) inlength was ligated and transformed into DH5acells. A recombinant named SasPET was identifiedwith RE and re-transformed into BL21 cells.SasPET was digested with EcoRI and XhoI, andthe foreign fragment was cloned into vector pGEX-6P-1. A positive recombinant identified with REwas named Sas6P-1. All the recombinants were se-quenced to confirm the authenticity of the se-quence.

3 Results

3. 1 Identification of SaPRO with RESaPRO was digested with RE according to the

physical map of the vector pProExHTb and Sa se-quence. The digested fragments were visualized by0.8% agarose gel electrophoresis. This result wasidentical to theoretical calculation (Figure 1) .

~ Figure 1. Identification of SaPRO with RELane 1: DL 15,000 DNA marker (TaKaRa, China).Lane 2: SaPRO digested with XhoI, of about 7.1 kb.Lanes 3 and 4: Vector pProExHTb linearized with EcoR I and

KpnI,of about 4.8 kb.Lanes 5 and 6: Fragment Sa digested with EcoR I and Kpn I, of

about 2.3 kb.

Lane 7: SaPRO digested with EcoR I and Kpn I, of about 2. 3kb and 4. 8 kb respectively.

Lane 8: DNA Ladder (MEI Fermentas) .

3.2 Identification of SaPETRecombinant SaPET was identified with RE

and primer-specific PCR. The agarose gel elec-trophoresis showed that Sa has been insertedpET30c vector (Figure 2) .

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Figure 2. Identification of recombinant SaPETLane 1: Identification of SaPET by ne.,ted PCR, of about 1.2 kb.

Lane 2: DL 15,000 DNA marker.

Lane 3: SaPET digested with EcoRI, of about 7.7 kb.Lane 4: SaPET digested with Xho I, of about 7. 7 kb.

3.3 Identification of SasPETSasPET was analyzed with RE, and the result

indicated that 5' end fragment of S gene about 1100bp in length has been cloned into pET30c (Figure3) .

Figure 3. Identification of recombinant SasPETLane 1: DL 15,000 DNA marker.

Lane 2: SasPET digested with EcoRI and XhoI,of 1. 1 kb and 5.4 kb re.-;pectively.

Lane 3: SasPET digested with EcoRI, of 6.6 kb.

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Life ScienceJournal, 3 (1 ) ,2006, Yin, et al, Constructionof Prokaryotic Expression VectorsBearing S Gene

3.4 Identification of Sas6P-lSas6P-1 was digested with RE, and the analy-

sis result of agarose gel indicated the correct inser-tion of fragment of interest (Figure 4) ,

3.5 Sequencing analysisThe sequencing relX>rtverified there was no

nucleotide insertion and deletion in the S gene usedin this study and it was identical to the publishedsequence (GenBank 1M accession numberAF494337) .

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Figure 4. Identification of recombinant Sas6P-lLane 1: DL 15,000 DNA marker.Lane 2: Sas6P-l digested with BamHI, of about 6.1 kb.Lane 3: Sas6P-l digested with cEcoRI, of about 6.1 kb.Lane 4: Vector pEGX-6P-llinearized with EcoRI and X/wI, of

about 4.9 kb.

4 Discussion

Gene expression system ( GES) is very usefulin genetic engineering field. Currently, prokaryot-ic, yeast and eukaryotic expression systems are fre-quently used GES. In which reformed E. coli isvery lX>pularfor protein production in large scaledue to their rapid growth rate, facilitation for con-tinuous fermentation and relatively low cost. How-ever, the genome of E. coli is well characterizedand a lot of cloning vectors are available (Grissham-mer, 1995).

The initial purlX>seof this study is to express5' end half of S gene using E. coli. However theexpression level of foreign gene in E. coli might beinfluenced by the type of prokaryotic expressionvector and length of gene of interest (Francisco,1993; Wels, 1995; lahn, 1995).

Therefore, we took full advantage of differentvectors and different fragments of S gene to con-struct various recombinant plasmids. At the sametime, we tried to decrease the length of S gene un-der the circumstance of retaining the major antigensites in order to increase the probability of success-ful expression of S gene. The usage of different

vectors enables us to make multiple choices for fu-ture protein purification.

In the process of construction, the sequencedrecombinants were utilized to give gene of interest,which enhanced the positive recombination ratioand decreased {X>SSiblemutation might derived fromPCR amplification. However, it should be notedthat open reading frame (ORF) of S gene must becorrect after inserting into suitable vectors to pre-vent the shift of ORF. Most vector we used hereare high efficient vectors that can express gene ofinterest in the form of fusion protein, decrease thetoxicity to host cells and facilitate the downstreampurification.

To sum up, we constructed four recombinantplasmids encoding the major antigenic sites ofTGEV. These lX>sitiveE. coli strains might be in-duced to express the gene of interest, which pro-vided the useful materials for further related re-search.

AcknowledgmentsThis work was partially sUPlX>rtedby funds

from China Scholarship Council(CSC) and by grantfrom "Project of the Tenth-five" of HeilongjiangProvince Scientific and Technique Committee, Chi-na.

Correspondence to:Yijing Li, Xiaofeng RenDepartment of VeterinaryCollege of Veterinary MedicineNortheast Agricultural University59 Mucai Street, HarbinHeilongjiang 150030, ChinaEmail: rxfemail@yahoo.com.cn

yijingli@yahoo. com

References

1. Francisco ]A, Campbell R, Iverson BL, et al. Productionand fluorescence-activated cell sorting of Escherichia coliexpressing a functional antibody fragment on the externalsurface. Proc Natl Acad Sci USA 1993;90:10444-8.

2. Gebauer F, Posthumus WP, Correa I, et al. Residues in-

volved in the antigenic sites of transmissible gastroenteri-tis coronavirus S glycoprotein. Virology 1991: 183 :225-38.

3. Grisshammer R, Tate CG. Overexpression of integralmembrane proteins for structural studies. Q Rev Biophys1995:28:315 - 422.

4. ]ahn S, Roggenbuck D, Niemann B, et al. Expression ofmonovalent fragments derived from a human IgM autoan-tibody in E. coli: The input of the somatically mutatedCDRI ICDR2 and of the CDR3 into antigen bindingspecificity. Immunobiology 1995:193:400-19.

5. Krempl C, Schultze B, Laude H,et al. Point mutationsin the S protein connect the sialic acid binding activity

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. 65 .

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p185erbB-2-specific single-chain antibody toxins differ intheir cell-killing activity on tumor cells expressing bothreceptor proteins. lnt J Cancer 1995;60: 137- 44.

9. Yin JC, Ren XF, Li YJ. Molecular cloning and phyloge-netic analysis of ORF7 region of Chinese isolate TH-98from transmissible gastroenteritis virus. Virus Genes2005;30:395 - 401.

Life Science Journal,3 (1) ,2006, Yin, et al,. Omstruction of ProkarYJtic Expression Vectors Bearing S Gene

I.

II

with the enteropathogenicity of transmissible gastroen-teritis coronavirus. J Viro11997; 71: 3285 - 7.

6. Ren XF, Liu BQ, Li YJ. Cloning and homology compar-ison of S gene for isolate TH-98 of porcine transmissiblegastroenteritis virus. Agric Sci China 2003; 2: 314 - 20.

7. Tuboly T, Nagy E, Dermis JR, et al. lmmunogenicityof the S protein of transmissible gastroenteritis virus ex-pressed in baculovirus. Arch Viro11994; 137: 55 - 67.

8. Wels W, Beerli R, Hellmann P, et al. EGF receptor and

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Received September 3 ,2005

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